Fabrication and Evaluation of a Sulfide Microelectrode for Biofilm

Chapter 19

Fabrication and Evaluation of a Sulfide Microelectrode for Biofilm Studies 1

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Downloaded by UNIV OF MICHIGAN ANN ARBOR on November 7, 2017 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/bk-1998-0690.ch019

Tong Yu , Paul L. Bishop , Ahmed Galal , and Harry B . Mark, Jr. 1

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Department of Civil and Environmental Engineering, University of Cincinnati, Cincinnati, OH 45221-0071 2Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172

In this chapter we describe the fabrication procedure and the evaluation of a solid-state ion-selective sulfide microelectrode for potentiometric measurement o f total dissolved sulfide i n aqueous solution at neutral p H . The sulfide microelectrode was made from a silver wire fused into a lead glass micropipette with a tip diameter o f 20 μ m . Its tip was chemically treated with HgCl solution before being sulphidized with alkaline N a S solution. The sulfide microelectrode exhibited similar characteristics to that o f the conventional sulfide electrode. The linear response o f the microelectrode is i n the range o f 5x10 to 5x10 M sulfide i n alkaline solution (pH=13.5). Its Nearnstian slope is - 31.1 m V per concentration decade. In neutral solution (pH 7.2) its linear response is i n the range o f 5x10 to 5x10 M total dissolved sulfide. Its slope i n this range is -51.2 m V per concentration decade. The p H effect on the response slope o f the sulfide microelectrode and the possibility o f sulfate interference were also examined. 2

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In this chapter we describe the fabrication and evaluation o f a solid-state ion-selective microelectrode for the determination o f total dissolved sulfide i n aqueous solution at neutral p H . The total dissolved sulfide, S , is designated i n this paper as the sum o f dissolved hydrogen sulfide ( H S ) , bisulfide (HS") and sulfide (S ~). The need for such a microelectrode derives from the study o f biological sulfate: reduction i n environmental biofilms, which typically have a neutral p H and a biofilm thickness about 1-3 m m . A microelectrode is needed to penetrate the biofilms to directly measure the concentration profiles o f sulfide. T

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©1998 American Chemical Society

Akmal and Usmani; Polymers in Sensors ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

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The commercially available sulfide electrode is too large for this application. It also requires the treatment o f samples with sulfide anti-oxidant buffer ( S A O B II), which raises the p H o f the samples to above 12 (7). Since the early 1980's, researchers i n microbial ecology have used sulfide microelectrodes to study microbial mats and biofilms (2-5). Their sulfide microelectrode was a solid-state A g / A g S ionselective microelectrode. The tip diameter o f the microelectrode was initially 200 μηι, which was later reduced to 20-50 μιη. It was used to measure biological samples at neutral p H . This microelectrode was made from a platinum wire fused into a lead glass capillary. The tip was ground flat and then recessed 30-50 μ ι η by etching i n a K C N solution. The recess was filled with silver by electroplating i n a solution containing A g N 0 and K C N . A layer o f A g S was formed on the surface o f the silver by dipping the electrode into an ( N H ) S solution. However, the microelectrode was stable only for several hours due to the conversion o f all the silver to A g S (2).


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In an effort to introduce and improve this microelectrode technique for environmental engineering biofilm research, we started our study o f sulfide microelectrode with the basic structure o f a modified Whalen's type oxygen microelectrode fabricated i n our laboratory (6-8). The potential advantages o f this microelectrode are its very small tip (approximately 5 μ ι η diameter) and the ease o f its fabrication. This microelectrode is made from a l o w melting-point (47°C) Bismuth alloy, consisting o f Bismuth (44.7%), Lead (22.6%), Indium (19.1%), T i n (8.3%), and Cadmium (5.3%) (9). The tip o f the microelectrode was chemically treated with or electroplated i n an ( N H ) S solution under various conditions. However, the experimental results indicated that the potential responses o f the electrode were not reproducible between measurements (10, 11). This lack o f reproducibility may be caused by the complexity and uncertainty o f the surface chemistry related with this alloy. 4

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Exploring a new direction, we tried to fabricate the sulfide microeletrode using the procedure described i n this paper, except for the last step. The pure silver tips o f the microelectrodes, with and without a recess etched i n a K C N solution, were chemically treated with 21.6% ( N H ) S solution for 2 min. Unfortunately, these microelectrodes did not respond very well to the concentration change o f the sulfide standard solutions even at alkaline p H . A l s o , we noticed under the microscope that a fluffy black substance formed at the tip o f the microelectrodes after repeated calibrations, possibly an indication o f the dissolution o f the silver (77). In the search for a better method to fabricate the sulfide microelectrode, we learned that Radie and M a r k had observed an interesting phenomenon i n their study o f a sulfide millielectrode (72). They noted that, for a pure silver (99.99% A g ) wire, no black sulfide layer on the surface was observed even after six days o f chemical treatment, and that only the silver wire containing 2.5-6.0% copper produced a silver sulfide layer after chemical treatment. They suggested that the silver sulfide layer is probably produced by an anodic reaction from a localized corrosion process. Dobcnik et. al. reported that the chemical pretreatment o f pure silver wire with H g also produced a silver sulfide layer on the surface and a good sulfide ion-selective electrode (75). 4

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In this study we: (a) developed a fabrication procedure for the sulfide microelectrode, (b) evaluated this procedure by calibrating the microelectrode at alkaline p H and by comparing it with a commercial sulfide electrode, (c) calibrated the


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sulfide microelectrode at the specified neutral p H range, and (d) analyzed the p H effect and examined possible interferences i n its application. The sulfide microelectrode made using this procedure has a tip diameter o f 20 μπι. It is suitable for direct potentiometric measurement o f sulfide concentrations i n the range o f 5 χ 10" to 5 χ 10" M i n alkaline solution, and o f total dissolved sulfide concentrations i n the range o f 7

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5 x 10" to 5 χ 10" M i n neutral solution. It can be used for the measurement o f sulfide i n the study o f biological sulfate reduction i n environmental biofilms.

Fabrication This sulfide microelectrode has a tip diameter o f approximately 20 μπι. It is made from a silver wire rather than a platinum one. The fine silver wire is etched i n a K C N solution and fused into a pulled glass micropipette. The tip o f the microelectrode is beveled, cleaned and chemically treated with first H g C l solution and then N a S solution. The use o f the silver wire makes the microelectrode body reusable after rebeveling the tip and repeating the chemical treatment as described i n the last step o f section Fabrication. In addition, the steps o f making the recess at the tip by etching i n a K C N solution and o f filling the recess with silver by electroplating (2) can be eliminated. There are two key steps i n the fabrication: first, to melt the glass such that it forms a good seal between the glass coating and the tip o f the silver wire, and second, to pretreat the beveled tip with H g C l solution before chemical treatment with the N a S solution. A l l o f the chemicals used i n this study were o f analytical grade. The fabrication procedures for the sulfide microelectrode is as follows: 2

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Pulling the Glass Micropipette. The lead glass micropipette (WPI, Catalog N o . PG10150-4) was chosen because o f its low softening point, good sealability and good insulating characteristics (14). The lead glass micropipette contains 22% P b O and has a softening point o f 625°C. The commercially available micropipette has an o.d. o f 1.5 m m , an i.d. o f 0.75 mm, and a length o f 10 cm. The center along the length o f the micropipette was heated over the flame o f a small burner until it became equally soft all the way around the cross-section at the heating point. This was done by constantly rolling the micropipette i n the outer end o f the flame between the fingers holding it. The micropipette was then taken quickly out o f the flame and instantaneously pulled from both ends to about twice o f its original length. A s a result, the middle section o f the micropipette became very fine but still unbroken. (

Etching the Silver Wire. The original pure silver (99.99% A g ) wire has a diameter o f 0.127 m m (Aldrich, Catalog N o . 26,555-1). The tip o f the wire was etched to approximately 10 μ ι η i n 2 M K C N solution (see section Potassium Cyanide Solution below). The setup is shown i n Figure 1. The silver wire served as one electrode while the graphite rod (from the core o f a N o . 2 pencil) served as the other. A voltage between 2-4 V (a.c.) was applied. M o s t o f the time, only the outermost 5-10 m m o f the silver wire was immersed i n the K C N solution and the wire was occasionally moved up and down to generate a smooth transition zone. The time at w h i c h the power is turned off is crucial to the final size o f the tip. It seems that the silver wire


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was etched away section by section i n the length o f several millimeters. W i t h good illumination, one can see with the eye exactly when the outermost tip disappears. The power should be turned off before the disappearance of the tip of the next section. The etched silver wire was then cleaned by immersing it sequentially i n three beakers o f M i l l i - Q water (18.2 cmQ). The whole process o f etching must be conducted i n a highly ventilated hood. Refer to the Material Safety Data Sheet o f K C N for safety precautions. Potassium Cyanide Solution. 2 M K C N solution was prepared by dissolving 6.512 g K C N i n 50 m l M i l l i - Q water. A few particles o f N a O H were added to the solution to keep it alkaline. Following the Material Safety Data Sheet o f K C N for safety precautions.

Inserting the Etched Silver Wire. The etched silver wire was put on a microelectrode holder (Narishige, Catalog N o . H - l ) attached to a micromanipulator (World Precision Instruments Catalog N o . M3301). The pulled glass micropipette was held horizontally by a clamp. The etched silver wire was very carefully inserted into the hollow center o f the pulled glass micropipette at one end until the tip o f the silver wire reached the very fine middle section o f the micropipette. This process was closely watched through two magnifiers at right angles to each other, as shown i n Figure 2. (There is only one magnifier showed in the figure.) It is essential that the fine tip is not bent during the insertion. Then the quality o f the tip can be examined under the microscope. Melting the Glass to Coat the Tip of the Silver Wire. The pulled glass micropipette with the etched silver wire inserted i n it was hung vertically, with the etched tip o f the silver wire pointing up. The glass micropipette was placed at the center o f a trough heating filament (Sutter Instrument C o . , Catalog N o . FT330B), as shown i n Figure 3. The heating filament was attached to a micromanipulator (World Precision Instruments Catalog N o . M3301) and connected to an adjustable voltage transformer (Scientific Products, Catalog N o . E2101-2). The heating filament was placed approximately several millimeters below the tip o f the silver wire. A small rubber stopper was fitted around the lower end o f the glass micropipette as a small weight. A small beaker with a piece o f sponge covering the bottom was placed underneath the glass micropipette. The heating operation was monitored carefully through a horizontal microscope (World Precision Instruments, Catalog N o . P Z M H ) . Heat was applied slowly by turning the voltage transformer gradually up until the glass started to slowly melt. Heat was continuously increased as slowly as possible until the tip o f the silver wire started to move downward. A t this moment, the heat was suddenly increased. The lower half o f the glass micropipette dropped into the beaker. The,lead glass should form a thin coating on the tip o f the silver wire and the tip should be sealed by the glass. It is crucial to slowly increase the voltage all the way through the heating operation. Turning the heat up suddenly at the last moment prevents the silver wire from breaking off before reaching the tip.



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Figure 1. Set-up for etching the silver wire.

Figure 2. Set-up for inserting the etched silver wire into the pulled glass micropipette.



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Figure 3. Apparatus and set-up for melting the glass to coat the tip o f the silver wire, a) A n overall view, b) Close-up: the heating filament and the tip.


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Beveling the Tip. The prepared glass micropipette containing the silver wire was placed on a micropipette beveler (Sutter Instrument C o . , Catalog N o . B V - 1 0 ) . The tip was beveled on the diamond abrasive plate with a 45° angle to expose the silver surface at the tip. The beveled tip was examined under a microscope. A good seal between the glass coating and the tip o f the silver wire is essential. A n alternative method is to examine under the microscope i f the silver surface at the tip is intact after the cleaning with H N 0 , as described i n the last step o f section Fabrication. A recess o f the silver at the tip after the quick H N 0 treatment usually indicates poor sealing (therefore, a much larger area for the silver to react with and to be corroded by the HN0 ). 3


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Assembling the Microelectrode. A small piece o f the l o w melting point Bismuth alloy (8) and then a fine electric wire were inserted into the glass micropipette from the tail end. The alloy was heated and melted to connect the silver wire and the electric wire. The electric wire was then connected to a B N C cable. The tail end o f the glass micropipette was sealed with epoxy resin (Elmer's). The connection to the B N C cable was sealed with electric connector sealant (Archer, Catalog N o . 278-1645). Chemically Treating the Tip. The purpose o f chemical treatment o f the tip is to form an ion-selective sensing layer. The chemical treatment includes three steps: (1) Clean the tip o f the microelectrode i n an ultrasonic water bath (Fisher Scientific, Catalog N o . 15-336) for approximately 20 seconds. Then treat it with 1:1 H N 0 . Finally, rinse it thoroughly with M i l l i - Q water. (2) Pretreat the tip with 0.1 M H g C l (see section Mercury Chloride Solution) for 15 minutes. Then rinse it thoroughly with M i l l i - Q water. (3) Treat the tip with 0.1 M alkaline solution o f N a S (see section Sodium Sulfide Solution) for another 15 minutes. Then rinse it thoroughly with M i l l i - Q water. Here, the chemical pretreatment o f H g is essential to form the sulfide sensing layer. The less volatile N a S , instead o f the ( N H ) S , was used to sulphidize the silver surface. Figure 4 shows the Scanning Electron Microscopic ( S E M ) pictures o f the tip of the sulfide microelectrode after the above treatment. The raised center is the sensing layer on top o f the silver surface. Surrounding it is the glass coating. The tip diameter o f the sulfide microelectrode is approximately 20 μ ι η . After the above treatment, the sulfide microelectrode was kept i n water before use. 3

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Mercury Chloride Solution. 0.1 M H g C l solution was prepared by dissolving 2.715 g H g C l i n 100 m l M i l l i - Q water. Sodium Sulfide Solution. The 0.1 M N a S solution was prepared as described i n Sulfide Standard Solutions at Alkaline p H under Evaluation. 2

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Evaluation In order to evaluate whether the above fabrication procedure produces a good sulfide microelectrode, the sulfide microelectrode was first calibrated against sulfide standards at alkaline p H (as required by the instruction manual o f the commercial sulfide electrode (7)). The calibration curve was compared with that o f the commercial sulfide electrode to see whether both exhibit similar electrode characteristics. Then



Figure 4. S E M pictures o f the tip o f the sulfide microelectrode. a) Front view: the raised center is the sensing layer formed on the surface o f the silver and the surrounding is the glass coating, b) Side view: the raised center is the sensing layer formed on the surface of the silver and the tip is beveled with a 45° angle.



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the microelectrode was examined for its electrode characteristics at neutral p H by calibrating against the total sulfide standards at the neutral p H . It was also compared with the commercial sulfide electrode calibrated at the same neutral p H . The result o f the calibration at alkaline p H demonstrates that the sulfide microelectrode has similar characteristics to that o f the commercial sulfide electrode and that the fabrication procedure used for making the sulfide microelectrode is valid. The result o f the calibration at neutral p H shows that the sulfide microelectrode is suitable for direct potentiometric measurement of total dissolved sulfide at neutral p H . Finally, the effect of p H on the electrode characteristics and the possible interference under the application were also examined. Calibration at Alkaline p H . The sulfide microelectrode was first calibrated at alkaline p H . The standard method D4685-92 o f the American Society for Testing and Materials ( A S T M ) (75) and the Orion Instruction Manual for Sulfide Electrode M o d e l 94-16 (7) were used as references. The sulfide microelectrode was calibrated against serial sulfide standard solutions at alkaline p H (see section Sulfide Standard Solutions at Alkaline pH). A A g / A g C l millielectrode (Microelectrodes Inc., Catalog N o . MI-409) was used as its reference electrode. The response o f the sulfide microelectrode was also compared with that o f the commercial sulfide electrode (Orion, Catalog N o . 9 4 1 6 B N , tip diameter 12 mm) and its A g / A g C l reference electrode (Orion, Catalog N o . 900200). A n Accumet Glass B o d y Combination p H Electrode (Fisher, Catalog N o . 13-620-285) was used to monitor the p H i n the standard solutions. These electrodes were connected to three Accumet Microprocessor M o d e l 15 p H / m V meters (Fisher, Catalog N o . 13-635-15A). These electrodes were immersed i n each beaker o f the sulfide standard solutions i n turn. Between measurements, the electrodes were rinsed with M i l l i - Q water and the commercially purchased electrodes were blotted dry. It was found that the sulfide microelectrode started to deteriorate after overnight storage, regardless o f whether it was stored i n 0.1 M N a S 0 , M i l l i - Q water, or i n air. Therefore, for each experiment i n this study, the step o f Chemically Treating the T i p in Fabrication, the preparation o f the standard solutions, and the calibration o f the sulfide microelectrode were all done on same day. We recommend that when measuring the concentration o f sulfide i n environmental biofilms the measurements should all be done on the same day as well. 2

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Figure 5 shows the calibrations o f the sulfide microelectrode and the commercial sulfide electrode at the alkaline p H . The upper curve is the calibration o f the sulfide microelectrode. The filled dots are the mean o f four sets o f experimental data, while the range bars indicate the maximum and minimum values. The straight line is the least square linear regression o f the four sets o f data. The linear response range o f the sulfide microelectrode is from 5 x l 0 " to 5 x l 0 " M , or from 0.016 to 160.3 mg/1 as S. Its slope, -31.1 m V per concentration decade, is near Nernstian, compared to the theoretical value o f -29.5 m V per concentration decade. The response time varied from 2 to 5 minutes. The p H values o f the standard solutions were between 13.5 and 13.6 (not shown i n the figure). The lower curve i n the figure is the calibration o f the commercial sulfide electrode. The filled squares are the mean o f five sets o f experimental data, while the range bars (barely seen) indicate the maximum and minimum values. The straight line is the least square linear regression o f the five sets 7

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of data. The linear response range o f the commercial sulfide electrode is from 1.125xl0" to 1.125xl0" M , or 0.0036 to 360.7 mg/1 as S. Its slope is -28.3 m V per concentration decade. The response time (90%) was less than 1 minute. The p H values o f the standard solutions were between 13.4 and 13.5 (not shown i n the figure). This calibration and comparison demonstrates that the sulfide microelectrode exhibits a similar slope and linear response range to that o f the commercial sulfide electrode. The noticeable difference i n the potentials between the sulfide microelectrode and the commercial sulfide electrode is likely due to the significant difference i n the nature of the ion exchange matrix. The sensing layer, with a diameter less than 20 μιη, at the tip of the sulfide microelectrode was generated i n situ by letting the A g wire chemically react first with H g C l and then with N a S . The sensing layer generated i n this way may have a relatively more porous structure and chemically different composition than that o f the commercial sulfide electrode. The sensing layer o f the commercial sulfide electrode was generated by first pressing A g S crystals, under high pressure, to a pellet which has a diameter about 10 m m and then fixing it to the tip o f the electrode. Therefore, the two electrodes have different sensing layers which indeed exhibit different electrode potentials. Sulfide Standard Solutions at Alkaline p H . Precise sulfide standard solutions can not be prepared by weighing the sulfide salt because o f the large and variable water o f hydration (75). Therefore, the following procedure was used: (a) A stock solution o f saturated sodium sulfide was prepared by dissolving approximately 100 g N a S - 9 H 0 i n 100 m l deaerated M i l l i - Q water. This solution was shaken, securely stoppered, and allowed to stand overnight. A N a S solution, when acidified, can release hydrogen sulfide which is extremely toxic, even at l o w levels. Therefore, when dealing with N a S solution, it is important to follow the Material Safety Data Sheet o f N a S for safety precautions, (b) A high concentration standard solution was prepared by first dissolving 16 g N a O H i n 100 m l deaerated M i l l i - Q water i n a 200 m l flask and allowing it to cool, then pipetting an exact amount (e.g. 10 ml) o f the stock solution prepared i n the previous step into the flask, and finally filling the flask to the mark using deaerated M i l l i - Q water. This standard solution contains approximately 0.1 M N a S and 2 M N a O H with a p H o f about 13.5. It was then titrated with 0.1 M P b ( C 1 0 ) (Orion, Catalog N o . 948206), using the commercial sulfide electrode (Orion, Catalog N o . 9416BN) as an indicator electrode, to determine its exact concentration o f sulfide. The high concentration standard solution can be kept for a week (7). This 0.1 M standard solution o f sulfide is also the 0.1 M N a S solution used to treat the tip o f the sulfide microelectrode as described i n section Fabrication, (c) The other standard solutions were prepared by serial dilution o f the high concentration standard solution using equal volumes o f 2 M N a O H and deaerated M i l l i - Q water. These standard solutions should be prepared with minimum aeration to avoid air oxidation o f sulfide i n the solutions. They should be prepared on the same day o f the microelectrode calibration.


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Calibration at Neutral p H . The p H has a significant effect on the chemical equilibrium o f sulfide. Environmental biofilms are usually not at alkaline p H values, but rather at near neutral values. A t neutral p H , the predominant species is no longer S " (16). But the total dissolved sulfide, S , designated i n this paper as the sum o f 2

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dissolved H S , HS", and S ", w i l l still be the same amount as the S " measured at alkaline p H . The approach used to calibrate the sulfide microelectrode at neutral p H was to determine the S " concentration o f the high concentration standard solution at alkaline p H and use this value as S at neutral p H . For biological sulfate reduction, the typical p H is 7.2 to 7.3 (5). Therefore, a phosphate buffer with p H 7.2 was used to prepare all standard solutions i n this calibration. The sulfide microelectrode was calibrated against the serial total dissolved sulfide standard solutions at the neutral p H (see section Total Dissolved Sulfide Standard Solutions at Neutral pH). The experimental set-up for the calibration at neutral p H was the same as that at alkaline pH. The sulfide microelectrode and its reference millielectrode, the commercial sulfide electrode and its reference electrode, and the combination p H electrode were placed together and then immersed i n turn i n each beaker containing the standard solutions. Between measurements, these electrodes were rinsed with M i l l i - Q water and the commercially purchased electrodes were blotted dry. 2

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Figure 6 is the calibrations o f the sulfide microelectrode and the commercial sulfide electrode at the neutral p H . The upper curve is the calibration o f the sulfide microelectrode. The filled dots are the mean o f two repeated experimental measurements, while the range bars (barely seen) indicate the maximum and minimum values. The straight line is the least square linear regression o f the two sets o f data. The linear response range o f the sulfide microelectrode at this p H is from 5 . 7 5 x 1 ο to 5.75xl0" M , or from 0.18 to 184.3 mg/1 as S. Its slope is -51.2 m V per concentration decade. Its response time (90%) was dependent on the total dissolved sulfide concentration and varied from 1 minute for the high concentrations to 2 to 5 minutes for the l o w concentrations. The p H values o f all the standard solutions were at 7.2, except for the 5.75xl0" M sample, where the p H changed to 7.4. (The p H data were not shown i n the figure). The lower curve i n the figure is the calibration o f the commercial sulfide electrode. The filled squares are the mean o f two repeated experimental measurements, while the range bars indicate the maximum and m i n i m u m values. The straight line is the least square linear regression o f the two sets o f data. The linear response range o f the commercial sulfide electrode at this p H is also from 5.75xl0" to 5 . 7 5 x l 0 ' M , or from 0.18 to 184.3 mg/1 as S. Its slope is -41.9 m V per concentration decade. Its response time is the same as that o f the sulfide microelectrode at neutral p H . The p H values o f the standard solutions were also the same as that of the sulfide microelectrode. -6

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The result o f this calibration and comparison shows that this sulfide microelectrode is suitable for measurement o f total dissolved sulfide i n aqueous solution at neutral p H . A t the specified neutral p H , the sulfide microelectrode performed as well as the commercial sulfide electrode did. When the concentration o f total dissolved sulfide was lower than 10" M , the sulfide microelectrode performed even better than the commercial sulfide electrode. A t the specified neutral p H , the slope o f the sulfide microelectrode, -51.2 m V per concentration decade, differs from the theoretical Nernstian slope, which is -29.5 and -59.0 m V per concentration decade for the dianion and monoanion reaction, respectively. These values indicate that the slope o f the sulfide microelectrode is somewhere between the theoretical Nernstian slopes o f the dianion and monoanion reactions, as would be expected as both anions are present at neutral p H . 5



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Figure 5. Calibrations o f the Sulfide Microelectrode and the Commercial Sulfide Electrode at Alkaline p H . The upper curve is sulfide microelectrode and the lower curve is commercial sulfide electrode. Experimental data are expressed as • and while is the corresponding regression.

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Figure 6. Calibrations o f the Sulfide Microelectrode and the Commercial Sulfide Electrode at Neutral p H . The upper curve is sulfide microelectrode and the lower curve is commercial sulfide electrode. Experimental data are expressed as · and • , while is corresponding regression.


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Total Dissolved Sulfide Standard Solutions at Neutral p H . A phosphate buffer was used to maintain the p H o f the standard solutions at 7.2. The buffer was prepared using N a H P 0 , N a H P 0 and M i l i - Q water (77) and was flushed with nitrogen gas continuously to deaerate it. The stock solution o f saturated sodium sulfide and the high concentration standard solution at alkaline p H were prepared i n the same way as described i n (a) and (b) i n section Sulfide Standard Solutions at Alkaline p H . Then, the high concentration standard solution at the neutral p H was prepared by first pipetting the exactly same amount o f the stock solution as used for the high concentration standard solution at alkaline p H into a 200 m l flask, and then filling the flask to the mark using the deaerated p H 7.2 phosphate buffer. This high concentration standard solution contains the exactly same amount o f total dissolved sulfide as that i n the high concentration standard solution at alkaline p H . The other standard solutions were prepared immediately by serial dilution o f the high concentration standard solution at the neutral p H using the deaerated p H 7.2 phosphate buffer. These standard solutions must be prepared just before the microelectrode calibration. The same caution should be applied to avoid air oxidation o f sulfide i n the solutions.


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The Effect of p H . To examine the effect o f p H , the sulfide microelectrode was calibrated at three different p H values i n the neutral range: 7.0, 7.2, and 7.4. These values were chosen because they are centered at the specified neutral p H (7.2) for this study and they can all be maintained by phosphate buffer (77). The method o f calibration was the same as that used i n section Calibration at Neutral p H . A l l calibrations at the three p H values were conducted on the same day. Figure 7 shows the effect o f p H on the calibration o f the sulfide microelectrode. The upper, middle and lower curves are the calibrations o f the sulfide microelectrode at p H 7.0, 7.2 and 7.4, respectively. The filled squares, dots and diamonds are the means o f two repeated experimental measurements at p H 7.0, 7.2 and 7.4, respectively. The range bars (barely seen for most points) indicate the maximum and minimum values. The straight lines are the least square linear regression o f the two sets o f data for each p H value respectively. Since the calibration at p H 7.4 started at 1.15xl0" M , for purposes o f comparison the three curves show only the linear response range from 1.15xl0" to 5.75x10" M . (The slope o f the calibration at p H 7.2 was therefore slightly different from that i n section Calibration at Neutral pH.) The slopes o f this linear response range o f the sulfide microelectrode are -55.4 m V per concentration decade for p H 7.0, -48.4 m V per concentration decade for p H 7.2, and -45.4 m V per concentration decade for p H 7.4. Table I summarizes the slopes o f the sulfide microelectrode at four different p H values. These experimental results reveal that the slope o f the sulfide microelectrode decreased from -31.1 to -55.4 m V per concentration decade while p H of the standard solutions decreased from 13.5 to 7.0. These intermediate slope values are exactly what would be predicted, assuming the use o f normally accepted p K values for H S and HS" (pK =7.02 and pK^ =13.9 (18)). For accurate measurement of total dissolved sulfide i n biofilm samples, the exact p H value o f the sample is a necessity. 5

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ρΗ=7.0 Υ = -55.4 Log(X) - 708 R = 0.993

< Downloaded by UNIV OF MICHIGAN ANN ARBOR on November 7, 2017 | http://pubs.acs.org Publication Date: April 17, 1998 | doi: 10.1021/bk-1998-0690.ch019

2

ρΗ=7.0 τ

> ε

ρΗ = 7.2 Υ = -48.4 Log(X) - 700 R = 0.996

ρΗ=7.2

2

ρΗ=7.4

.2 -500

ρΗ = 7.4 Υ = -45.4 Log(X) - 703 R = 0.997 2

1e-5

1e-4

1e-3

Concentration of Total Sulfide (M) 0.1

1

10

100


Figure 7. Effect o f p H on the Calibration o f Sulfide Microelectrode. The • and the upper curve are for the calibration at p H 7.0. The · and the middle curve are for the calibration at p H 7.2. The • and the lower curve are the calibration at p H 7.4. Experimental data are expressed as • , · and • while regression.

Table I.

is corresponding

Slopes of the Sulfide Microelectrode at different p H Values

Dianion Nearnstian reaction pH=13.5 p H = 7.4 p H = 7.2 p H = 7.0 Monoanion Nearnstian reaction

Slope o f Linear Response Range ( m V per concentration decade) -29.5 -31.1 -45.4 -51.2 -55.4 -59.0


245

Interference Study of Sulfate. Although most reported interfering species for sulfide electrodes (1, 12, 13) are not present at a significant level i n biological sulfate reduction, sulfate, i n equilibrium with sulfide, is abundant i n the system. In order to examine whether sulfate interferes with the measurement o f sulfide, the fixed interference method (19) was used. The sulfide microelectrode was calibrated i n serial standard solutions at p H 7.2 with a fixed concentration o f N a S 0 . The N a S 0 , after being dried at 103°C overnight, was added to the p H 7.2 phosphate buffer and its concentration was fixed at 5 x l 0 ' M . The method o f calibration was the same as that used i n section Calibration at Neutral pH, except the sequence o f measurement was from l o w concentration to high concentration standard solutions and then, without rinsing, repeated immediately from high concentration to l o w concentration standard solutions. The purpose is to examine whether there is concentration hysteresis on the sulfide microelectrode. The experimental results were compared with that o f the calibration at p H 7.2 without N a S 0 . Figure 8 shows the calibration o f the sulfide microelectrode at p H 7.2 with and without N a S 0 . The filled dots are the means o f the two repeated experimental measurements at p H 7.2 with 5 x l 0 " M o f N a S 0 , while the filled squares are the experimental measurements reported i n section Calibration at Neutral p H ( p H 7.2 without N a S 0 ) . The upper straight line is the least square linear regression of the calibration without N a S 0 (the filled squares). Its slope is -51.2 m V per concentration decade. The lower straight line is the least square linear regression o f the calibration with 5χ10" M o f N a S 0 from 1.04xl0" to 5.20x10" M o f total dissolved sulfide. Its slope is -50.8 m V per concentration decade. The two lines are very close from approximately l x l O " to 5 x l 0 " M total dissolved sulfide. Considering that the two calibrations used two different sulfide microelectrodes and were conducted on two different days, the difference is within the 2

4

2

4


3

2

4

2

4

3

2

2

4

4

2

4

3

5

2

4

3

5

3

Sulfide Microelectrode pH=7.2 Without Na S0 Y = -51.2 Log(X)-709 R = 0.99

*

2

•

4

2

Sulfide Microelectrode pH=7.2 With 0.005 M N a S 0 Y = -50.8 Log(X)-713 R = 0.98 2

£ -

6 0 0

4

2

1e-6

1e-5

1e-4

1e-3

Concentration of Total Sulfide (M) I

•

0.1

1

1

1

10

Ί —

100


Figure 8. Calibration o f the Sulfide Microelectrode at p H 7.2 with and without N a S 0 . The • and the upper straight line are the experimental data and the regression for the calibration without N a S 0 . The · and the lower straight line are the experimental data and the regression for the calibration with N a S 0 . 2

4

2

4

2


4

246

experimental error. Therefore, we conclude that sulfate does not interfere with the sulfide measurement using the sulfide microelectrode under these experimental conditions. This conclusion is also agree with the result o f Dobcnik et. al. on their study o f a sulfide millielectrode (75). A s showed by the range bars o f the filled dots i n Figure 8, no concentration hysteresis was noticed with the sulfide microelectrode from 1.04xl0" to 5.20x10" M of total sulfide. 5

3


Conclusions The fabrication procedure described i n this chapter produces a practical solid-state ionselective sulfide microelectrode. The microelectrode has a tip diameter o f 20 μ ι η . It was made from a silver wire melted into a lead glass micropipette. Its tip was beveled and then chemically treated with H g C l solution before being sulphidized with N a S solution to form a sensing layer. The sulfide microelectrode can be used for potentiometric measurement o f sulfide. A t alkaline p H (pH >13.5), the sulfide microelectrode exhibits similar characteristics to that o f the commercial Orion sulfide electrode. The linear response range o f the sulfide microelectrode is from 5 x l 0 ' to 5 x l 0 " M sulfide, or from 0.016 to 160.3 mg/1 as S. Its Nearnstian slope is - 31.1 m V per concentration decade. The sulfide microelectrode is also suitable for measuring total dissolved sulfide (designated as the sum o f H S , HS" and S ") i n aqueous solution at neutral p H . A t p H 7.2, the linear response range o f the sulfide microelectrode is from 5.75x10" to 5.75x10" M total dissolved sulfide, or from 0.18 to 184.3 mg/1 as S. Its slope i n this range is -51.2 m V per concentration decade. 2

2

7

3

2

2

6

3

The p H has a significant effect on the characteristics o f the sulfide microelectrode. The result o f this study reveals that when p H decreased from 13.5 to 7.0 the microelectrode slope o f the linear response range decreased from -31.1 to -55.4 m V per concentration decade. A t neutral p H , the slope is typically i n between the Nearnstian slope o f a dianion and a monoanion reaction (29.5 and 59.0 m V per concentration decade, respectively). For accurate measurement o f total dissolved sulfide at neutral p H , it is necessary to know the exact p H value. Sulfate, i n equilibrium with sulfide i n biological sulfate reduction, does not interfere with the sulfide measurement.

Acknowledgment This study was supported by the National Institute o f Environmental Health Sciences, N I H and E P A (Grant number P42 E S 04908). Its contents are solely the responsibility of the authors and do not necessarily represent the official views o f the N I E H S , N I H and E P A . The authors thanks Dr. W i l l i a m R. Heineman for valuble discussions.


247

References

1. 2. 3.


4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Orion Model 94-16 Silver/Sulfide Electrode Instruction Manual. Revsbech, N. P.; Jørgensen, Β. B.; Blackburn, T. H. Limnol. Oceanogr. 1983, 28, 1062-1074. Revsbech, N. P.; Jørgensen, Β. B. In Advances in Microbial Ecology; Marshall, K.C.,Ed.; Plenum Press: New York, London, 1986; Vol. 9, pp. 293-352. Nelson, D.C.;Jørgensen, Β. B.; Revsbech, N. P. Appl. Environ. Microbiol. 1986, 52, 225-233. Kühl, M.; Jøgensen, Β. B. Appl. Environ.Microbiol.1992, 58, 1164-1174. Whalen, W. J.; Riley, J.; Nair, P. J.Appl.Physiol.1967, 23, 798-801. Bungay, H. R., 3rd; Whalen, W. J.; Sanders, W. M. Biotech. Bioeng. 1969, 11, 765-772. Fu, Y.-C. Ph.D. Dissertation, University of Cincinnati, Cincinnati, Ohio, 1993. Belmont Alloy 2451 In Alloy Digest; Engineering Alloy Digest, Inc.: Upper Montclair, New Jersey, 1978; p. Bi-3. Galal, Α.; Yu, T.; Bishop, P. L.; Mark, J., Harry B. Presented at Pittcon'96, Chicago, Illinois, March 3-8, 1996. Yu, T. Unpublished Experimental Data. Radie, Ν.; Mark, Harry B., Jr. Croatica Chemica Acta 1985, 58, 179-188. Dobcnik, D.; Gomiscek, S.; Stergulec, J. Fresenius J.Anal.Chem. 1990, 337, 369-371. World Precision Instruments, 1995 Catalog, p. 55. ASTM D4658-92. Snoeyink, V. L.; Jenkins, D. Water Chemistry; John Wiley & Sons: New York, 1980; Chapter 4, pp. 86-196. Gerhardt, P.; Murray, R. G. E.; Wood, W. Α.; Krieg, N. R., Eds. Methods for General and Molecular Bacteriology; American Society for Microbiology: Washington, D.C., 1994; Chapter II, pp. 135-294. Martell, A. E.; Smith, R. M. Critical Stability Constants; Plenum Press: New York, 1974. Kluka. Selectophore: Ionophores, Membranes, Mini-ISE; Kluka Chemical Corp.: Switzerland, 1996, p. 11.


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